The effect of embryo extract on the types of collagen synthesized by cultured chick chondrocytes.

Growth of embryonic chick chondrocytes in dialyzed embryo extract results in both a change in morphology of the cells toward that of a fibroblast and a change in the type of collagen synthesized from the cartilage-specific Type II collagen (chain composition [α1(II)]₃) to a mixture of Type I collagen (chain composition [α1(I)]₂α2) and the Type I trimer (chain composition [α1(I)]₃). Analyses after 6 days of growth in embryo extract show that the synthesis of only Type I collagen and the Type I trimer can be detected. However, on subculturing the cells to a low density and allowing a period of growth without embryo extract, colonies of chondrocytes reappear and the synthesis of Type II collagen apparently resumes. It is suggested that the observed changes represent a “modulation” in cell behavior, this being expressed not only by the morphological changes but also by changes in cell-specific protein synthesis as demonstrated by the changes in the type of collagen synthesized.     

In Vivo Administration of a JAK3 Inhibitor during Acute SIV Infection Leads to Significant Increases in Viral Load during Chronic Infection

The studies reported herein are the first to document the effect of the in vivo administration of a JAK3 inhibitor for defining the potential role of NK cells during acute SIV infection of a group of 15 rhesus macaques (RM). An additional group of 16 MHC/KIR typed RM was included as controls. The previously optimized in vivo dose regimen (20 mg/kg daily for 35 days) led to a marked depletion of each of the major NK cell subsets both in the blood and gastro-intestinal tissues (GIT) during acute infection. While such depletion had no detectable effects on plasma viral loads during acute infection, there was a significant sustained increase in plasma viral loads during chronic infection. While the potential mechanisms that lead to such increased plasma viral loads during chronic infection remain unclear, several correlates were documented. Thus, during acute infection, the administration of the JAK3 inhibitor besides depleting all NK cell subsets also decreased some CD8⁺ T cells and inhibited the mobilization of the plasmacytoid dendritic cells in the blood and their localization to the GIT. Of interest is the finding that the administration of the JAK3 inhibitor during acute infection also resulted in the sustained maintenance during chronic infection of a high number of naïve and central memory CD4⁺ T cells, increases in B cells in the blood, but decreases in the frequencies and function of NKG2a⁺ NK cells within the GIT and blood, respectively. These data identify a unique role for JAK3 inhibitor sensitive cells, that includes NK cells during acute infection that in concert lead to high viral loads in SIV infected RM during chronic infection without affecting detectable changes in antiviral humoral/cellular responses. Identifying the precise mechanisms by which JAK3 sensitive cells exert their influence is critical with important implications for vaccine design against lentiviruses.     

Type IV collagen from chicken muscular tissues. Isolation and characterization of the pepsin-resistant fragments

Type IV collagen has been isolated from adult chicken gizzard after limited pepsin digestion and subsequent differential salt fractionation in acidic and neutral conditions. After denaturation, three fragments (called F1, F2, and F3) were isolated by agarose gel filtration and carboxymethylcellulose chromatography. F1 and F2 possessed apparent molecular weights of 53 000 and 50 000, respectively, and were consistently isolated in a 2:1 proportion. F3 was larger and after reduction of disulfide bonds gave rise to three fragments (called F3A, F3B, and F3C) of apparent molecular weights 68 000, 40 000, and 29 000. No alpha-chain-sized components of Type IV collagen were observed. A native fraction containing F1 and F2, but no F3, was isolated after extraction using less pepsin and an additional salt fractionation in acidic conditions. F1 and F2 in the native form were not separated by carboxymethylcellulose or diethylaminoethylcellulose chromatography performed in nondenaturating conditions or by differential salt precipitation in acidic or neutral conditions; these results suggest that F1 and F2 arise as a single native component of structure (F1)2F2. The fraction containing F1 and F2 also gave rise to a single segment long spacing crystallite pattern and to a circular dichroism spectrum which was typical for a native collagen. F1 and F2 were also isolated from chicken heart, blood vessels, and skeletal muscle, whereas from bovine aorta, using the same isolation procedures, two alpha-chain-sized components were obtained, which appeared to be similar to the two Type IV chains recently described by other groups. The data suggest that (i) pepsin fragmentation of type IV collagen from chicken tissues occurs in a different manner compared to Type IV collagen from mammalian tissues and (ii) for the chicken there must be at least two Type IV chains which are assembled into a single native molecule.     

Connective-tissue macromolecules in Golgi chicken tendon organs and at their interface with muscle fibers and adjoining tendinous structures.

Tendon organs from leg and forearm muscles of white leghorn chickens were examined with a library of monoclonal antibodies to determine the composition of their connective-tissue framework and the types of connective-tissue macromolecules that occur at the sites where muscle fibers attach to the receptors. The capsules of the tendon organs were positive for connective-tissue macromolecules typical of basal lamina (collagen type IV, laminin, and heparin sulfate proteoglycan) and for tenascin, collagen types III and VI, and fibronectin. Connective-tissue bundles in the lumen of a receptor reacted primarily with antibodies against collagen type I and 4-chondroitin sulfate. The narrow partitions that divide each lumen into compartments stained for collagen type III. Toward its tendinous end, a receptor made few contacts with muscle fibers. Instead, the capsule and the collagenous bundles blended gradually with the intermuscular portions of tendons. At the muscular end, the connections were more complex. Muscle fibers that attached in series to tendon organs split to produce basal lamina-covered, finger-like extensions, which were separated from each other by fissures. Tongues of connective tissue containing tenascin, collagen types I and VI, and fibronectin extended into the fissures. Distally the tongues were continuous with the tenascin in the capsule and just internal to the capsule, fibronectin and basal lamina macromolecules in the capsule, and collagen type I in the collagenous bundles. The uninterrupted presence of these macromolecules around terminating muscle fibers and in the capsule and/or the intraluminal collagen bundles suggests that muscle fibers that attach in series at the muscular end exert a force during muscular contraction on the intraluminal collagen bundles and on the receptor capsule.